CC0651 is an allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme that potently inhibits the ubiquitination of p27Kip1 (IC50: 1.72 μM).

p27Kip1 ubiquitination:1.72 μM

CC0651 functions by stabilizing a weak interaction between ubiquitin and the E2 donor ubiquitin-binding site. It completely inhibits polyubiquitin chain assembly, reduces the formation of free triubiquitin, and minimally affects hCdc34 monoubiquitin production, with no impact on diubiquitin production [1]. CC0651 significantly impairs the initiation rate of ubiquitin chains on substrates by SCFCdc4, as indicated by the monoubiquitination of Sic1 by K0 ubiquitin. Interestingly, CC0651 enhances the formation of ubiquitin dimers and monoubiquitinated hCdc34, consistent with the accumulation of the hCdc34 conjugate in cells treated with its ester derivative. A quantitative SCF ubiquitination assay using a β-Catenin substrate peptide determined an IC50 value of 18±1 μM for CC0651 inhibition, aligning with concentrations effective in NMR and TR-FRET assays [2].